Chapter 9: Laboratory Diagnosis

Clinical microbiology laboratories play a fundamental and indispensable role in providing reliable and timely information regarding the identification of infectious disease agents. Clinicians use this information not only to make or confirm a diagnosis, but also to guide clinical decisions and treatment options, so this information needs to be definitive, significant, and relevant to the case under consideration. Obtaining accurate results that can be interpreted with high confidence is dependent on the laboratory receiving high-quality patient specimens.

Since specimen selection and collection are typically the responsibility of the medical staff, clinicians should (1) understand the pathogenesis of the infection and ensure proper collection of an adequate quantity of specimen from the body site that is most likely to yield growth of the infecting organism, while avoiding contamination from the normal flora; (2) ensure that the integrity of the specimen is not compromised during transport and that the specimen is handled in such a way to preserve the viability of any anaerobes or fastidious organisms; and (3) provide ancillary information to guide the laboratory personnel who will process and analyze the specimen.

As diagnostic microbiology testing becomes more complex, clear communication and strong partnerships between microbiology lab professionals and clinicians will remain a top priority. Clinicians communicate crucial clinical information about a patient to microbiology lab staff that will then allow lab personnel to direct clinicians toward context-appropriate tests and optimized sample collection methods that have the most diagnostic value, which will ultimately lead to better outcomes for patients.

Traditionally, diagnosis has relied on culture, microscopic and phenotypic characterization of an organism (Table 9–1), and serologic testing, in which the organism is identified by the detection of organism-specific antibodies in the patient’s serum. More recently, advances in the fields of molecular biology and genomics have resulted in more accurate and rapid identification of causative. There are now several US Food and Drug Administration (FDA)-approved nucleic acid and proteomic-based assays for identifying infectious agents (discussed below), and their routine implementation in the clinical laboratory setting has resulted in improved patient care, better antibiotic stewardship resulting in decreased antimicrobial resistance rates, and increased efficiency of the laboratory and healthcare facility in the processing and analysis of clinical samples.

Microscopic Examination

If a specimen is collected from a “sterile” body site that does not harbor a “background” of normal flora (e.g., sterile tissues, cerebral spinal fluid, joint fluid, or urine), a sample of the specimen can be prepared for microscopic examination using an appropriate staining method, like the Gram stain or acid-fast stain. If bacteria are seen in the specimen, their shape (e.g., cocci or rods), size, and arrangement (e.g., chains or clusters) and whether they are gram-positive, gram-negative, or acid-fast should be noted and can be useful in their identification. It is also important to determine whether only one or more than one type of bacteria is present. The microscopic appearance is typically not sufficient to definitively identify an organism, but often allows an educated guess to be made regarding the taxonomic classification (genus) of the organism and thus guides empiric therapy that can be initiated without waiting for growth of the organism.

Culture-Based Methods

Several methods for diagnosing bacterial or fungal infections require the suspected pathogen to be isolated in pure culture from a properly obtained clinical specimen. Historically this has been accomplished by using an agar-based medium, for instance, blood agar plates, and streaking the specimen over the agar surface in a manner to obtain well-isolated colonies. The agar plates are then incubated under atmospheric conditions that will support the growth of a variety of different microorganisms, including those that are most likely to be causing the patient’s symptoms based on the clinical evidence. The media can be “selective,” containing compounds that only allow certain bacteria to grow (e.g., antibiotics, salts, or dyes), and/or “differential,” because they contain other compounds that allow one type of bacteria to be distinguished from another based on a biochemical reaction (e.g., detecting hemolysis on blood agar plates or pigment formation). Table 9–2 contains a list of various bacteriologic agars commonly used in the diagnostic laboratory and the function of these agars. Once pure, well-isolated colonies are obtained, further phenotypic characterization (e.g., analyzing biochemical and enzymatic activities) and antibiotic susceptibility testing (see Chapter 11) can be performed.

In modern Clinical Microbiology Laboratories, this process of cultivation has been improved upon substantially with the development and introduction of novel nylon flocked swabs (Eswabs) and accompanying transport containers. The Eswabs have significantly improved pathogen recovery rates and allow for the elution of >90% of sample organisms into liquid transport media as opposed to agar-base transport media.

There are several advantages to liquid-based cultivation. Having high recovery of a homogenous distribution of organisms in the transport medium facilitates the use of automated plating instruments, allows for many more plates to be inoculated from the same clinical specimen, and has shifted the practice of microbiology from labor-intensive, tubed biochemical identification and susceptibility testing methods to a more accurate and efficient automated sample processing pipeline using plates and assays that can be incubated, monitored, and read automatically. A substantial body of research has shown that automation of specimen processing and incubation has decreased institutional costs, technician errors, and time-to-specimen identification, all of which should improve on patient care and safety.

Since specimen selection and collection are of paramount importance in yielding high-quality laboratory results, common specimen collection sites and methods are discussed below.

Blood Cultures

Blood cultures are performed most often when sepsis, endocarditis, osteomyelitis, meningitis, or pneumonia is suspected. The bacteria most frequently isolated from blood cultures are two gram-positive cocci, Staphylococcus aureus and Streptococcus pneumoniae, and three gram-negative rods, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Certain pathogenic fungi including yeast (Candida species and Cryptococcus neoformans) and molds can also be isolated from blood cultures.

For blood cultures, the site for venipuncture must be cleansed with an antiseptic to prevent contamination by members of the flora of the skin, usually Staphylococcus epidermidis, and decrease the risk of infection-related complications. The blood obtained is added to a rich growth medium in a bottle that contains an indicator for carbon dioxide (CO₂) production. Standard practice is to inoculate 10 mL of blood into each of two bottles per culture set, with one bottle incubated aerobically and one anaerobically. Production of CO₂ within the bottle indicates that organism metabolism and growth have occurred. Once growth occurs, Gram stain, subculture, and antibiotic sensitivity tests are performed. In some hospitals, molecular methods are used to identify the organism (see later in this chapter).

Throat Cultures

Throat cultures inoculated onto blood agar plates continue to be used to detect the presence of group A β-hemolytic streptococci (Streptococcus pyogenes or GAS), an important and treatable cause of pharyngitis. They are also used when diphtheria, gonococcal pharyngitis, or thrush (Candida) is suspected.

In the past several years, the FDA has approved point of care (POC) devices that test for GAS antigens (Rapid Antigen Detection Tests [RADT]) and, most recently, a molecular diagnostic test based on detection of GAS-specific DNA sequences (see a general discussion of nucleic acid molecular testing in the Molecular Diagnostic Methods section below). This nucleic acid amplification test (NAAT) is much more sensitive and specific than the RADT, and can detect fewer than 50 bacterial cells/mL of GAS from a throat swab in as little as 15 minutes, which is significantly faster than the 24–hours required for plate cultivation of the specimen followed by bacitracin susceptibility testing to confirm GAS infection.

Since accurate diagnosis and appropriate antimicrobial therapy is recommended to prevent post-GAS infection complications (rheumatic fever or acute glomerulonephritis), POC testing is preferable to traditional plate culture. The rapid definitive diagnosis of infection facilitates initiation of definitive antimicrobial therapy, which aligns with institutional efforts on antimicrobial stewardship to improve quality of care.

Whether the specimen is being obtained for plate culture or POC testing, obtaining a high-quality specimen is important. The collection swab should touch not only the posterior pharynx, but also both tonsils or tonsillar fossae as well. The sample can them be inoculated onto a blood agar plate and streaked to obtain single colonies, or can be used to inoculate the POC devices. If cultivating on blood agar plates, GAS will form β-hemolytic colonies after 24 hours of incubation at 35°C. Further testing looking at bacitracin susceptibility (traditional) or MALDI-TOF analysis (described below) can be used to determine whether the organism is likely to be a group A Streptococcus.

Note that a Gram stain is typically not done on a throat swab because it is impossible to distinguish between the appearance of the normal flora streptococci and S. pyogenes.

Sputum Cultures

Sputum cultures can be performed to determine infectious etiologies of pneumonia or to test for active pulmonary tuberculosis. The most frequent bacterial cause of community-acquired pneumonia is S. pneumoniae, whereas S. aureus and gram-negative rods, such as K. pneumoniae and P. aeruginosa, are common causes of hospital-acquired pneumonias.

It is important that the specimen for culture really be sputum and not saliva or nasopharyngeal secretions from the upper airway. Examination of a Gram-stained smear of the specimen frequently reveals whether the specimen is satisfactory. A reliable specimen has more than 25 leukocytes and fewer than 10 epithelial cells per 100× field. An unreliable sample can be misleading and should be rejected by the laboratory. If the patient cannot cough and the need for a microbiologic diagnosis is strong, tracheal aspirate, bronchoalveolar lavage, or lung biopsy may be necessary. Because these procedures bypass the normal flora of the upper airway, they are more likely to provide an accurate microbiologic diagnosis. A preliminary assessment of the cause of the pneumonia can be made by Gram stain if large numbers of typical organisms are seen.

Culture of the sputum on blood agar can reveal the presence of colonies, with identification established using various serologic or biochemical tests or by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (see below). Cultures of Mycoplasma are infrequently done; diagnosis is usually confirmed by a rise in antibody titer. If Legionella pneumonia is suspected, the organism can be cultured on charcoal-yeast agar, which contains the high concentrations of iron, sulfur, and cysteine required for growth.

If active tuberculosis is suspected, an acid-fast stain can be done to look for the organism in the sputum; however, this microscopic method is not very sensitive. The sputum can also be cultured on special media, but requires at least 6 weeks of incubation to cultivate Mycobacterium tuberculosis. There is now a NAAT (Xpert MTB/RIF assay) available to detect M. tuberculosis infection. This test, which can be completed in less than 2 hours, not only confirms the presence of M. tuberculosis, but also detects whether the infecting strain is resistant to rifampin.

Aspiration pneumonia and lung abscesses represent other types of infections affecting the respiratory tract. If these are suspected, culturing for anaerobic bacteria is important.

Cerebrospinal Fluid Cultures

Cerebrospinal fluid (CSF) cultures can be performed primarily when a neurologic infection such as meningitis, meningoencephalitis, or transverse myelitis is suspected. CSF specimens from tissue-centric cases, including encephalitis, brain abscess, and subdural empyema, may show negative cultures. The most important causes of acute bacterial meningitis are three encapsulated organisms: Neisseria meningitidis, S. pneumoniae, and Haemophilus influenzae.

Because acute meningitis is a medical emergency, the specimen should be taken immediately to the laboratory. The Gram-stained smear of the sediment of the centrifuged sample guides the immediate empirical treatment. If meningitis caused by acid-fast bacteria such as M. tuberculosis is suspected, an acid-fast stain and culture of CSF should be performed, although for the reasons discussed above, nucleic acid amplification methods should also used for rapid identification.

The fungus C. neoformans, a cause of meningitis, particularly in human immunodeficiency virus–infected patients, can also be cultured from CSF. In the past, the India ink test was performed, but at present, most laboratories use the latex agglutination test for C. neoformans (cryptococcal antigen) done on CSF as a more specific and sensitive test.

Stool Cultures

Most cases of acute diarrhea are self-limiting and require neither empiric antimicrobial therapy nor stool culture. However, testing will be performed for patients experiencing severe or persistent diarrhea, for those with symptoms consistent with invasive disease (enterocolitis), patients who are immunocompromised, outbreak-associated diarrhea, and healthcare associated diarrhea. While Norovirus is the most common overall cause of diarrhea in the United States, the most common bacterial pathogens causing diarrhea are Salmonella, Shigella, Campylobacter, and E. coli O157 strains (STEC). Clostridium difficile should be suspected for patients who develop nosocomial diarrhea, particularly after antibiotic treatment. The patient’s stool can be tested for the presence of the C. difficile toxins using an enzyme immunoassay or for the presence of a toxigenic C. difficile strain using a NAAT.

When bacterial culture is recommended, feces collected during the acute phase of symptoms is the specimen of choice. Specimens should be processed by the clinical lab within 2 hours of collection to maximize detection of the organisms. The selection of primary plating media used for routine culture varies from laboratory to laboratory, but typically includes (1) MacConkey agar; (2) a selective/differential medium (e.g., eosin-methylene blue agar [EMB]) to maximize recovery of Salmonella and Shigella; (3) a medium to recover Campylobacter (e.g., Campy-CVA [cefoperazone, vancomycin, and amphotericin] or Skirrow medium), which must be incubated in a microaerobic atmosphere (5% O₂, 10% CO₂, and 85% N₂) at 42°C; and (4) a medium to recover E. coli O157, like MacConkey-sorbitol medium. Antigen detection assays to test for Shiga toxin I and II or C. difficile toxin A/B should also be performed.

MacConkey and EMB agars are both selective and differential. They are selective because they allow gram-negative rods to grow but inhibit many gram-positive organisms. Their differential properties are based on the fact that Salmonella and Shigella do not ferment lactose, whereas many other enteric gram-negative rods do. On EMB agar, colonies of E. coli, a lactose fermenter, appear purple and have a green sheen. In contrast, colonies of non-lactose fermenters, such as Salmonella and Shigella, appear colorless.

More and more clinical labs have incorporated MALDI-TOF technology (discussed below in the Molecular Diagnostics section) into their diagnostic tool repertoire, which is the preferred culture-based diagnostic method both from a cost and time-effective perspective. This method compares mass spectral libraries representing thousands of reference strains with a patient isolate to make sensitive and reliable identification of the unknown bacterial or fungal pathogen within minutes of sample preparation. Both from a cost and time-effective perspective, it is becoming the culture-based diagnostic test of choice.

It is important to note, however, that this technology cannot distinguish between all important pathogens. Currently, MALDI-TOF cannot accurately differentiate among all species within certain groups of organisms, such as the Enterobacter cloacae complex, Burkholderia cepacia complex, and Streptococcus bovis species group. In addition, Shigella and E. coli cannot be reliably differentiated by MALDI-TOF. Since E. coli is one of the most frequent organisms encountered in clinical microbiology laboratories, alternative strategies such as employing TSI (Triple Sugar Iron) slants or EMB agar to determine lactose fermentation would be necessary to distinguish E. coli from Shigella species. Organisms that fail to produce H₂S, and produce acid but not gas in the butt, and have an alkaline slant on TSI agar, would be subjected to slide agglutination by specific Shigella antisera to arrive at an identification.

There are also several culture-independent diagnostic tests (CIDTs) now available for suspected GI pathogens, which are based on immune assays to detect toxins, or nucleic acid amplification to detect pathogens. These tests are more expensive than culture-based testing, but they are highly sensitive and specific, and typically increase detection of all bacterial GI pathogens, especially those that are harder to cultivate (i.e., Campylobacter and Shigella). The main advantages of CIDTs are the speed at which a diagnosis can be obtained (hours versus days for cultivation) and their reliability. CIDTs should be confirmed by culture especially if susceptibility testing is indicated or if there is a suspected outbreak that would require a Public Health investigation.

Urine Cultures

Urine cultures are performed primarily when pyelonephritis or cystitis is suspected. By far the most frequent cause of urinary tract infections is E. coli. Other common agents are Enterobacter, Proteus, and Enterococcus faecalis.

Urine in the bladder of a healthy person is sterile, but it acquires organisms of the normal flora as it passes through the distal portion of the urethra. To avoid these organisms, a midstream specimen, voided after washing the external orifice, is used for urine cultures. In special situations, suprapubic aspiration or catheterization may be required to obtain a specimen. Because urine is a good culture medium, any organisms present in the specimen can multiply, leading to erroneous results regarding type and number of organisms present at the time of collection. Thus, it is essential that the cultures be done within 1 hour after collection or stored in a refrigerator at 4°C for not more than 18 hours.

It is commonly accepted that a bacterial count of at least 100,000/mL must be found to conclude that significant bacteriuria is present (in asymptomatic persons). There is evidence that a bacterial count as low as 1000/mL is significant in symptomatic patients. For this determination to be made, quantitative or semiquantitative cultures are performed. There are several techniques: (1) a calibrated loop that holds 0.001 mL of urine can be used to streak the culture; (2) serial tenfold dilutions can be made and samples from the dilutions streaked; and (3) a screening procedure suitable for the physician’s office involves an agar-covered “paddle” that is dipped into the urine—after the paddle is incubated, the density of the colonies is compared with standard charts to obtain an estimate of the concentration of bacteria.

Genital Tract Cultures

Genital tract cultures can be performed on specimens from individuals with an abnormal discharge or on specimens from asymptomatic contacts of a person with a sexually transmitted disease. One of the most important pathogens in the genital tract is Neisseria gonorrhoeae. The laboratory diagnosis of gonorrhea can be made by microscopic examination of a Gram-stained smear and culture of the organism, but is now typically done with nucleic acid techniques. Culture may still be important to determine antimicrobial susceptibility in cases of treatment failure.

Specimens are obtained by swabbing the urethral canal (for men), the cervix (for women), or the anal canal (for men and women). A urethral discharge from the penis is frequently used. Because N. gonorrhoeae is very delicate, the specimen should be inoculated quickly onto medium such as a Thayer-Martin chocolate agar plate.

Gram-negative diplococci found intracellularly within neutrophils on a smear of a urethral discharge from a man have over 90% probability of being N. gonorrhoeae. Because smears are less specific when made from swabs of the endocervix and anal canal, cultures or nucleic acid-based testing are necessary. The finding of only extracellular diplococci suggests that these Neisseriae may be members of the normal flora and that the patient may have nongonococcal urethritis.

Nongonococcal urethritis and cervicitis are also extremely common infections. The most frequent cause is Chlamydia trachomatis, which cannot grow on artificial medium but must be grown in living cells. For this purpose, cultures of human cells or the yolk sacs of embryonated eggs are used. The finding of typical intracytoplasmic inclusions when using Giemsa stain or fluorescent antibody is diagnostic. Because of the difficulty of culturing C. trachomatis, nonbacteriologic methods, such as NAAT, are now typically used to diagnose sexually transmitted diseases caused by this organism.

Because Treponema pallidum, the agent of syphilis, cannot be cultured, diagnosis is made primarily by serology and sometimes by microscopy if dark-field microscopy is available. The presence of motile spirochetes with typical morphologic features seen by dark-field microscopy of the fluid from a painless genital lesion is sufficient for the diagnosis. The serologic tests fall into two groups: (1) the nontreponemal antibody tests such as the Venereal Disease Research Laboratory (VDRL) or rapid plasma reagin (RPR) test and (2) the treponemal antibody tests such as the fluorescent treponemal antibody-absorption (FTA-ABS) test. These tests are described in Chapter 24.

Wound & Abscess Cultures

A variety of different organisms have been described in association with wound and abscess infections, and many of these infections are polymicrobial. The bacteria most frequently isolated differ according to anatomic site and predisposing factors. Abscesses of the brain, lungs, and abdomen are frequently caused by anaerobes such as Bacteroides fragilis and gram-positive cocci such as S. aureus and S. pyogenes. Members of the soil flora such as Clostridium perfringens are important causes of traumatic open-wound infections, while surgical-wound infections are commonly associated with skin flora including various staphylococci and streptococci and Propionibacterium acnes. Infections of dog or cat bites are often due to Pasteurella species (~50% of cases), whereas human bites usually involve viridans streptococci, especially Streptococcus anginosus, and mouth anaerobes, such as Prevotella and Fusobacterium.

Anaerobes are frequently involved in these types of infection, so it is important to place the specimen in anaerobic collection tubes and transport it promptly to the laboratory. Because many of these infections are due to multiple organisms, including mixtures of anaerobes and nonanaerobes, the specimen should be cultured on several different media under different atmospheric conditions. The Gram stain can provide valuable information regarding the range of organisms under consideration.

Sometimes an organism is not recovered by culturing, either because it is nonculturable on bacteriologic media or it is intermittently present or only present in limited numbers, and other techniques must be used. Table 9–3 describes some approaches to making a diagnosis when the cultures are negative, which include immunologic and molecular methods discussed below.

Serologic Methods

These methods are described in more detail in Chapter 64. However, it is of interest here to present information on how serologic reactions aid the microbiologic diagnosis. There are essentially two basic approaches: (1) using known antibody to identify the microorganism, and (2) using known antigens to detect antibodies in the patient’s serum.

Identification of an Organism with Known Antiserum

Slide Agglutination Test—Antisera can be used to identify Salmonella and Shigella by causing agglutination (clumping) of the unknown organism. Antisera directed against the cell wall O antigens of Salmonella and Shigella are commonly used in hospital laboratories. Antisera against the flagellar H antigens and the capsular Vi antigen of Salmonella are used in public health laboratories for epidemiologic purposes.

Latex Agglutination Test—Latex beads coated with specific antibody are agglutinated in the presence of the homologous bacteria or antigen. This test is used to determine the presence of the capsular antigen of the yeast C. neoformans.

Enzyme-Linked Immunosorbent Assay—In this test, a specific antibody to which an easily assayed enzyme has been linked is used to detect the presence of the homologous antigen. Because several techniques have been devised to implement this principle, the specific steps used cannot be detailed here (see Chapter 64). This test is useful in detecting a wide variety of bacterial, viral, and fungal infections.

Fluorescent Antibody Tests—A variety of bacteria can be identified by exposure to known antibody labeled with fluorescent dye, which is detected visually in the ultraviolet microscope. Various methods can be used, such as the direct and indirect techniques (see Chapter 64).

Identification of Serum Antibodies with Known Antigens

Slide or Tube Agglutination Test—In this test, serial twofold dilutions of a sample of the patient’s serum are mixed with standard bacterial antigen suspensions. The highest dilution of serum capable of agglutination is the titer of the antibody. As with most tests of a patient’s antibody, at least a fourfold rise in titer between the early and late samples must be demonstrated for a diagnosis to be made. This test is used primarily to aid in the diagnosis of typhoid fever, brucellosis, tularemia, plague, leptospirosis, and rickettsial diseases.

Serologic Tests for Syphilis—The detection of antibody in the patient’s serum is frequently used to diagnose syphilis, because T. pallidum does not grow on laboratory media. There are two kinds of tests.

1. The nontreponemal tests use a cardiolipin–lecithin–cholesterol mixture as the antigen, not an antigen of the organism. Cardiolipin (diphosphatidylglycerol) is a lipid extracted from normal beef heart. Flocculation (clumping) of the cardiolipin occurs in the presence of antibody induced by infection by T. pallidum. The VDRL and RPR tests are nontreponemal tests commonly used as screening procedures. They are not specific for syphilis but are inexpensive and easy to perform.

2. The treponemal tests use T. pallidum as the antigen. The two most widely used treponemal tests are the FTA-ABS and the T. pallidum particle agglutination (TPPA) tests. In the FTA-ABS test, the patient’s serum sample, which has been absorbed with treponemes other than T. pallidum to remove nonspecific antibodies, is reacted with nonviable T. pallidum on a slide. Fluorescein-labeled antibody against human immunoglobulin G (IgG) is then used to determine whether IgG antibody against T. pallidum is bound to the organism. In the TPPA test, the patient’s serum sample is mixed with gelatin particles that have been sensitized with T. pallidum whole cell antigens. Patient serum that contains antibodies to T. pallidum will react with the gel particle, resulting in agglutination that will appear like a uniformly distributed smooth mat of particles in the microtiter plate. A negative result appears as a compact button at the bottom of the microtiter plate.

Cold Agglutinin Test—Patients with Mycoplasma pneumoniae infections develop autoimmune antibodies that agglutinate human red blood cells in the cold (4°C) but not at 37°C. These antibodies occur in certain diseases other than Mycoplasma infections; thus, false-positive results can occur.

Molecular Diagnostic Methods

The field of molecular diagnostics is dynamic and rapidly evolving. The methods described below, and in some cases mentioned above, have been adopted by clinical microbiology labs not only due to their increased sensitivity and specificity and reduced turnaround time, as compared with more traditional diagnostics described above, but also because early and accurate diagnosis has a significant and positive impact on patient care. It is not the intent here to discuss all assays that are currently available or under development. Rather, it is important for clinicians to be cognizant of the pace of development of molecular techniques in the realm of clinical microbiology and to consult with the clinical microbiology lab when considering assays that are most appropriate for any given patient.

Genomic Tests

Molecular diagnostic tests can be broadly categorized into those that evaluate nucleic acids (DNA or RNA) and those that assay for proteins or enzymatic activity. There are three types of nucleic acid–based tests used in the diagnosis of bacterial diseases: NAATs, nucleic acid probes, and nucleic acid sequence analysis; many of these tests have become a routine part of clinical microbiology diagnostics. Nucleic acid–based tests can be performed rapidly, are highly specific and quite sensitive (especially the amplification tests), and can often be performed directly from the clinical specimen, mitigating the need to wait for culture results. They are therefore especially useful for those bacteria that are difficult to culture, such as Chlamydia and Mycobacterium species.

NAATs use polymerase chain reaction (PCR) or other amplifying process to increase the number of bacteria-specific DNA or RNA molecules so the sensitivity of the test is significantly higher than that of unamplified tests. Contemporary assays can target a single pathogen or use multiplexed panels containing multiple targets to identify pathogens associated with particular clinical syndromes (e.g., pneumonia, endocarditis, or meningitis).

As mentioned above, examples of FDA-approved NAAT tests include C. trachomatis and N. gonorrhoeae in urine samples in sexually transmitted diseases, the Luminex respiratory virus panel for detection of seven different respiratory viruses, and the BioFire FilmArray meningitis/encephalitis (ME) panel, which simultaneously detects 14 common infectious agents in CSF.

Tests that use nucleic acid probes are designed to detect bacterial DNA or RNA directly (without amplification) using a labeled DNA or RNA probe that will hybridize specifically to the bacterial nucleic acid from a cultured organism. These tests are simpler to perform than the amplification tests but are less sensitive.

Nucleic acid sequence analysis of ribosomal RNA (rRNA) can be used to identify bacteria or fungi. This is considered a universal approach because it is based on amplification of genes that are highly conserved within a given organism type, such as the 16S and 23S rRNA genes in bacteria and the 28S rRNA and ITS genes in fungi. A bacterium that had never been previously cultured, Tropheryma whipplei, was identified using this approach.

An emerging technique in diagnostic microbiology is metagenomic sequencing analysis. This shotgun approach aims to comprehensively identify infectious agents by randomly amplifying and sequencing all of the DNA and RNA in clinical samples. The typically small fraction of “nonhost” (nonhuman) sequences corresponding to potential pathogens is then mapped to large reference databases, such as the National Center for Biotechnology Information (NCBI) GenBank, to identify sequences from any virus, bacterium, fungus, or parasite that is present. Currently, this test is only available on a limited basis from a few select laboratories, such as the University of California, San Francisco Clinical Microbiology Laboratory, although tests based on this approach are rapidly being developed.

Many of these assays are often performed in concert with other culture-dependent methods; these approaches only confirm the presence of a nucleic acid target and do not prove the presence of a viable organism. In addition, the exquisite sensitivity of nucleic acid amplification–based methods makes it challenging to ensure that results are due to the actual presence of the target organism, and not postcollection contamination.

Proteomic Tests

Molecular proteomic platforms have also been developed, and one that is now being used in many clinical laboratories is MALDI-TOF mass spectrometry. MALDI-TOF technology measures particles based on their mass-to-charge ratio. In this technique, organisms that have been cultured and purified from clinical samples (bacteria and some types of fungi have been successfully analyzed) are embedded in a matrix material that, when excited by a laser, transfers charge from the matrix to the microbial macromolecules (proteins and nucleic acids) causing desorption of the newly ionized particles. These charged particles are then separated by their mass-to-charge ratio, yielding a mass spectral signature that is unique to a specific genus and often to the species level. Using bioinformatics, these MALDI-TOF spectra can be compared to standardized databases, and those that are highly aligned are identified as a match with a stated level of confidence. This assay, which takes less than a minute to complete once the sample is loaded into the machine, has been shown to be highly accurate, efficient, and cost-effective.

Despite the emergence of a diverse array of molecular and biochemical diagnostic tools, culture-based approaches to diagnosing infectious disease remain a mainstay of the clinical microbiology lab. A combination of new methodologies and classic techniques is central to the successful and accurate identification of microorganisms encountered in the clinical setting.